DNA double-strand breaks (DSB) can arise by ionizing radiation, alkylation damage, and replication, including improper processing of lagging strand intermediates. DNA breaks can be a powerful source of chromosome instability as well as programmed genetic modification. Cells have elaborate systems for dealing with DSBs, including DNA repair and checkpoint arrest to increase the opportunity for repair. DSBs in chromosomes lead to a checkpoint arrest at the G2/M boundary in yeast, which provides further opportunities for repair. DSBs are repaired through homologous recombination, end-joining , and by single-strand annealing at homologous regions beyond the breaks. Nearly all organisms exhibit these repair processes as well as checkpoint arrests. Defects in these processes are often associated with disease in humans including cancer. DNA ends must be processed to allow homologous interactions for recombination and single strand annealing. Endjoining involves only local nuclease degradation that enables interaction at microhomologies of only a few bases, The Ku and RAD50/MRE11/XRS2 (R/M/X) complexes of proteins are required for endjoining. In addition, the R/M/X complex functions in the nuclease processing of ends to provide recombination substrates. The Ku complex, which associates at the ends of breaks prevents excessive processing of broken ends as well as providing end joining. We are examining the consequences of DSBs in various mutants and the mechanisms of handling DSBs. Our approaches have been extended to consider repair in the context of chromosomes and chromatin organization. While DNA is the central component of chromosomes, there is little information about the relationship between DNA break repair, repair systems and chromatin organization. We have also continued studies on the characterization of genes that affect the sensitivity to a variety of double-strand breaking agents. Chromatin functions in repair and recombination. Assembly of new chromatin during S phase requires the histone chaperone complexes CAF-1 (Cac2p, Msi1p and Rlf2p) and RCAF (Asf1p plus acetylated histones H3 and H4). Cells lacking CAF-1 and RCAF are hypersensitive to DNA damaging agents such as methyl methanesulfonate and camptothecin, suggesting a possible defect in double-strand break (DSB) repair. Assays developed to quantitate repair of defined, cohesive-ended break structures revealed that DSB-induced plasmid:chromosome recombination was reduced approximately 10-fold in RCAF/CAF-1 double mutants. Recombination defects were similar with both chromosomal and plasmid targets in vivo, suggesting that inhibitory chromatin structures were not involved. Consistent with these observations, ionizing radiation-induced loss of heterozygosity (LOH) was abolished in the mutants. NHEJ repair proficiency and accuracy were intermediate between wildtype levels and those of NHEJ-deficient yku70 and rad50 mutants. The defects in NHEJ, but not homologous recombination, could be rescued by deletion of HMR-a1, a component of the a1/alpha2 transcriptional repressor complex. The findings are consistent with the observation that silent mating loci are partially derepressed when chromatin assembly is reduced. These results suggest a post-replicative repair function for CAF-1 and RCAF in recombination, possibly involving deposition of new histone octamers after DNA synthesis associated with strand exchange. Identification of genes required for resistance to double-strand breaking agents. The cellular response to DNA damaging agents involves many genes and pathways. We have taken a systematic approach to identifying all genes that impact on survival/growth response to ionizing radiation. In order to identify new recombination or checkpoint genes that are required for the maintenance of genetic integrity following induction of DSBs, we previously examined 3670 nonessential genes for the consequences of diploid homozygous mutations on growth and/or lethality following a single acute dose of IR. We initially found 107 new genes that were required for radiation resistance. Many of these appear to affect replication, recombination and checkpoint functions and >50% share homology with human genes including 17 implicated in cancer. We have now completed the study and a total of 169 new genes that are required for radiation toleration. Thus ~4% of the nonessential genes are required for toleration of IR damage. With the completion of this screen, we have determined for the first time the total complement of nonessential genes required for the toleration of IR damage. Approximately 90% of the genes affect resistance to other DNA damaging agents including bleomycin, doxorubicin, methyl methanesulfonate (MMS), hydroxyurea (HU), camptothecin and ultraviolet light (UV). Using existing genetic and proteomic databases, many of these genes were found to interact in a damage response network with the transcription factor Ccr4, a core component of the CCR4NOT and PAF-CDC73 transcription complexes. Deletions of individual members of these two complexes render cells sensitive to the lethal effects of IR as diploids, but not as haploids, indicating that the diploid G1 cell population is radiosensitive. Consistent with a role in G1, diploid ccr4 cells irradiated in G1 show enhanced lethality when compared to cells exposed as a synchronous G2 population. In addition, a prolonged RAD9-dependent G1 arrest occurred following IR of ccr4 cells and CCR4 is a member of the RAD9 epistasis group thus confirming a role for CCR4 in checkpoint control. Moreover, ccr4 cells that transit S phase in the presence of the replication inhibitor hydroxyurea (HU) undergo prolonged cell cycle arrest at G2 followed by cellular lysis. This S phase replication defect is separate from that seen for rad52 mutants since rad52 ccr4 cells show increased sensitivity to HU when compared to rad52 or ccr4 mutants alone. These results indicate that cell cycle transition through G1 and S phases is CCR4-dependent following radiation or replication stress.